German Cockroach Allergen Research Articles

Down-Modulation of Cockroach (CR) Allergen-specific Th2 Cell Responses Following Subcutaneous German Cockroach Allergen Immunotherapy (SCIT)

The responses of T cells to subcutaneous allergen immunotherapy (SCIT) are not fully elucidated. We conducted a functional immunological evaluation of cockroach (CR) allergen-specific CD4+ T cell reactivity in the double-blinded, placebo-controlled, multi-center CRITICAL study.

Changes in Prevalence Patterns of Allergen Sensitization During the COVID-19 Pandemic in South China: A Cross-Sectional Study from 2017 to 2020

Background: Public health measures during COVID-19 have led to an unprecedented change in social lifestyle which might have an impact on the allergen sensitization in population. We sought to explore the prevalence patterns of serum allergen-specific IgE (sIgE) sensitization and serum total immunoglobulin E (tIgE) level among patients with clinical symptoms of suspected allergic diseases before and during the COVID-19 pandemic in south China. Methods: We assessed cross-sectional datasets from the First Affiliated Hospital of the Guangzhou Medical University from 2017 to 2020. Data on clinical symptoms of the patients with suspected allergic diseases in the hospital were collected and data from February to June in each year were extracted. Chi-square test and Fisher exact test were used to test statistical significance among years. Adjusted odds ratios and 95% confidence intervals were used to assess the magnitudes of the differences among years. Findings: The number of hospital visits for patients with suspected allergy symptoms decreased compared with previous years during COVID-19. For inhalant allergens, there was a statistically significant difference (PDermatophagoides pteronyssinus, 1·43 (P=0·024) for Dermatophagoides farina, 1·50 (PDermatophagoides pteronyssinus and tIgE were statistically different (PInterpretation: During the COVID-19 pandemic, the positive rate of inhalant allergens and tIgE level increased while the positive rate of food allergens remained stable. Staying at home for a long time without frequent ventilation, insufficient sunshine or storing food at home might contribute to the result. Funding: The Science and Technology Development Fund, Macau SAR and the National Natural Science Foundation of China.Declaration of Interests: We declare no competing interest exists. Ethics Approval Statement: The study was approved by the ethic committee of the First Affiliated Hospital of Guangzhou Medical University (Reference number: GYFYY-2016-73) of Guangzhou Medical University. Written informed consent was obtained from all adults and legal guardians of the participating children.

Measurement of German cockroach allergens and their isoforms in allergen extracts with mass spectrometry.

Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non-standardized. More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency. To expand the multiple reaction monitoring (MRM) assay for an in-depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts. We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM-MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform-specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables. Evaluation of the commercial extracts confirmed the variability of production lots and commercial sources. Commonly used defatting and extraction protocols yielded extracts with comparable allergen profiles and content. However, the identity and quality of source materials was a major contributor to variability. In comparing commercial GCr extracts to environmental samples, relative quantities of Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 were similar, while Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were present in the environmental samples but largely absent for the commercial extracts. LC/MRM-MS can be used to measure all known GCr allergens in commercial allergen extracts and environmental samples. Significant differences exist between allergen profiles of commercial extracts and the profiles of environmental samples from dwellings. This analytical platform can serve as a template to achieve better product characterization of similarly complex products.

New German cockroach allergens contribute to IgE and T cell reactivity

New German cockroach allergens contribute to IgE and T cell reactivity

Optimal conditions for the storage of German cockroach extract

Allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease‑rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50%glycerol in NS, 0.3%phenol in NS, or 0.3%phenol and 50%glycerol in NS. The extracts in different buffers were stored either at room temperature (18‑26˚C, RT) or refrigerated (2‑8˚C). Subsequently, the protein concentration and allergen content (Blag1 and Blag2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Blag1, 65.2% Blag2, and 91.4%potency remained after one year when 50%glycerol NS was added to the extract with refrigeration. However, less than 13.7%protein, 17.1%Blag1, 0% Blag2 and 32.5% potency were maintained after one year when 50%glycerol NS was not added to the extract and was maintained at RT. The addition of 0.3%phenol NS did not show significant effects on extract stability. The addition of 50%glycerol NS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease‑rich cockroach extract.

Multiplex assay for high throughput potency and stability measurement of all known German cockroach (GCr) allergens: A step toward full characterization

Multiplex assay for high throughput potency and stability measurement of all known German cockroach (GCr) allergens: A step toward full characterization

Accurate quantification of 5 German cockroach (GCr) allergens in complex extracts using multiple reaction monitoring mass spectrometry (MRM MS).

German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy. The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC-MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts. We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high-resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD<10%), linear over a wide range (r>0.99, 0.01-1384fmol/μL), and sensitive (LLOD and LLOQ <1fmol/μL). Having established the parameters for LC-MRM MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy. Our data demonstrate that the LC-MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies.

Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2.

Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma.

Multiple Reactions Monitoring (MRM) Mass Spectrometry for Absolute Quantification of Allergens in German Cockroach (GCr) Allergen Extracts

Mass spectrometry (MS) offers a unique approach for rapid compositional characterization and absolute quantification of individual allergens and allergen isoforms in complex allergen extracts, and thus has the potential to transform the ongoing efforts of allergen standardization. We developed an assay to evaluate GCr extracts, based on liquid chromatography (LC) and MRM MS, by systematically optimizing the digestion conditions (buffer, protein denaturation, digestion time, and trypsin quantity), LC gradient, and MS parameters (collision energy, dwell time, precursor-fragment pairs). In silico simulation, followed by recombinant-allergen and extract digestion and high resolution MS analysis allowed us to identify 47 proteotypic and quantotypic reference peptides for use as calibrators and internal standards for quantification of endogenous peptides. The peptide list represents 9 allergens and 8 iso-allergens and variants. Since the initial phase of the project aimed to generate a template-method using selected GCr allergens, twenty-six peptides (13 native and 13 isotopically-labelled) representing Bla g 1, 2, 3, 4 and 5 were chemically synthesized and used in LC-MRM/MS method development. Robustness and suitability of the overall protocol was extensively assessed following the ICH (Q2(R1) and FDA guidelines. The protocol offers excellent precision (CV < 10%), linear response (r > 0.99), dynamic range (>3), detection, and recovery. The applicability of the method was further demonstrated using eight commercial GCr extracts from four different vendors. The LC-MRM/MS method proved to be a useful tool for absolute quantification of allergens in GCr extracts and has promise for the standardization of complex allergen extracts.

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

BackgroundGerman cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.ObjectiveOur aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.MethodsSingle chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA.ResultsChicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target.ConclusionsAn scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.

Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts

BackgroundRecent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. ObjectiveTo assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. MethodsMixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. ResultsModerate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. ConclusionAspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal–insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections.

Bla g 3: a novel allergen of German cockroach identified using cockroach-specific avian single-chain variable fragment antibody

BackgroundThe IgE response to cockroach allergens is thought to be associated with asthma. German cockroach (GCr) allergen extract is a complex mixture of allergens, and the identification and characterization of immunodominant allergens is important for the effective diagnosis and treatment of GCr-induced asthma. ObjectiveTo characterize a novel GCr allergen homologous to the American cockroach allergen Per a 3. MethodsGCr-specific avian monoclonal antibodies were used for direct immunoprecipitation of specific targets from whole-body GCr extract. Precipitated protein was identified by mass spectrometry and sequence analysis. Putative recombinant protein also was expressed, purified, and used for determination of allergenicity, determined by IgE enzyme-linked immunosorbent assay with serum from 61 GCr-allergic patients. The identified target also was analyzed for heat stability using a bead-based assay. ResultsThe immunoprecipitated target of monoclonal antibody 2A1 was identified as a novel allergen of GCr homologous to American cockroach allergen Per a 3. This homolog, designated Bla g 3, has an apparent mass of 78 kDa, can be measured in GCr extract using antibody 2A1, and is a heat-stable protein. Screening of 61 serum samples from GCr-allergic patients showed a 22% prevalence of Bla g 3-specific IgE. ConclusionBla g 3 is a GCr allergen with structural homology to American cockroach allergen Per a 3.

Alveolar macrophages play a key role in cockroach-induced allergic inflammation via TNF-α pathway.

The activity of the serine protease in the German cockroach allergen is important to the development of allergic disease. The protease-activated receptor (PAR)-2, which is expressed in numerous cell types in lung tissue, is known to mediate the cellular events caused by inhaled serine protease. Alveolar macrophages express PAR-2 and produce considerable amounts of tumor necrosis factor (TNF)-α. We determined whether the serine protease in German cockroach extract (GCE) enhances TNF-α production by alveolar macrophages through the PAR-2 pathway and whether the TNF-α production affects GCE-induced pulmonary inflammation. Effects of GCE on alveolar macrophages and TNF-α production were evaluated using in vitro MH-S and RAW264.6 cells and in vivo GCE-induced asthma models of BALB/c mice. GCE contained a large amount of serine protease. In the MH-S and RAW264.7 cells, GCE activated PAR-2 and thereby produced TNF-α. In the GCE-induced asthma model, intranasal administration of GCE increased airway hyperresponsiveness (AHR), inflammatory cell infiltration, productions of serum immunoglobulin E, interleukin (IL)-5, IL-13 and TNF-α production in alveolar macrophages. Blockade of serine proteases prevented the development of GCE induced allergic pathologies. TNF-α blockade also prevented the development of such asthma-like lesions. Depletion of alveolar macrophages reduced AHR and intracellular TNF-α level in pulmonary cell populations in the GCE-induced asthma model. These results suggest that serine protease from GCE affects asthma through an alveolar macrophage and TNF-α dependent manner, reflecting the close relation of innate and adaptive immune response in allergic asthma model.

Allergen stabilities and compatibilities in mixtures of high-protease fungal and insect extracts

BackgroundCurrent practice guidelines state that protease-rich fungal and insect extracts can be combined when preparing immunotherapy vaccines, but data supporting the stability of allergens in these mixtures have not been reported. ObjectiveTo determine the stabilities and compatibilities of Alternaria alternata and German cockroach allergens in mixtures with other high-protease fungal and insect (cockroach, imported fire ant) extracts at final extract concentrations consistent with injection dose targets for maintenance immunotherapy. MethodsMixtures containing Alternaria, German cockroach, and other fungal and insect extracts frequently included in immunotherapy vaccines were analyzed by a combination of quantitative analyses (enzyme-linked immunosorbent assays for multiallergen immunoglobulin E [IgE]-binding potency, major Alternaria allergen Alt a 1, and major German cockroach allergens Bla g 1 and Bla g 2) and qualitative methods (immunoblotting). Mixtures and analogous single-extract controls containing 10 to 50% glycerin were evaluated after storage for up to 12 months at 2°C to 8°C. ResultsMixtures of extracts within the same phylogenetic groups (fungal-fungal, insect-insect) retained favorable Alternaria and German cockroach allergen levels and activities under most conditions examined. For several cross-taxonomic (fungal–insect) extract combinations at 10 to 25% glycerin concentrations, different immunochemical test methods measuring single (major) or multiple allergens yielded threefold to 10-fold variations in allergen recoveries. ConclusionAllergen compatibilities can be compromised in some fungal–insect extract mixtures, contrary to current immunotherapy practice parameter recommendations. Separation of these products into different treatment vials may be required to produce stable mixtures for subcutaneous immunotherapy. Data from assay methodologies with distinct binding specificities provide a critical assessment of allergen activities in high-protease extract mixtures.

Prevention and treatment of DNA vaccine encoding cockroach allergen Bla g 1 in a mouse model of allergic airway inflammation

One-fourth of the US population is sensitized to the German cockroach. Primary German cockroach allergen Bla g 1 is detected in 63% of homes and 52% of childcare facilities in the United States. No effective treatment or vaccination strategies are yet available. We evaluated the prophylactic and therapeutic efficacy of a plasmid DNA-mediated vaccination using the Bla g 1 gene in a mouse model of allergic inflammatory airway disease. A plasmid DNA vector coding for the Bla g 1 allergen controlled by cytomegalovirus promoter was constructed. To estimate the protective efficacy, BALB/c mice were given three injections of plasmid DNA-Bla g 1 prior to sensitization with two priming doses of recombinant Bla g 1 (rBla g 1) antigens, followed by nebulized rBla g 1 challenge. In the therapeutic approach, sensitization was followed by administering Bla g 1 DNA vaccine. Bla g 1 vaccination significantly reduced allergen-induced airway inflammation, even after mice were presensitized and a Th2-dominant response was established. The Bla g 1 vaccination significantly reduced total inflammatory cell infiltrate, eosinophilia, secretion of Th2 cytokines IL-4 and IL-5 in bronchoalveolar lavage fluid, allergen-induced inflammatory infiltrates in the lungs, and Bla g 1-specific IgE in serum upon challenge with rBla g 1. Importantly, Bla g 1 DNA vaccination was able to induce IL-10-secreting regulatory T cells that could suppress the allergen-specific Th2 cells. DNA vaccination showed protective and therapeutic efficacy against a clinically relevant allergen Bla g 1.

The Influence of Environmental Temperature and Humidity on Temporal Decomposition of Cockroach Allergens Bla g 1 and Bla g 2 in Feces

The aim of the study was to establish a model of the environmental fate of German cockroach (Blattella germanica L.) allergens Bla g 1 and Bla g 2 in feces under controlled and field conditions. Temporal decline (3, 6, and 9 mo) of allergens Bla g 1 and Bla g 2 in the feces protected from cleaning was measured under laboratory and experimental household conditions. The influence of environmental temperature (15, 20, 25, 30, and 35 degrees C) and moisture (53, 75, 85, and 100% RH) on allergen degradation was estimated for 3, 6, and 9 mo. Bla g 1 was more stable than Bla g 2 and the proteins. The proteins and Bla g 2 contents were correlated negatively with the decomposition time; Bla g 1 was not. However, when the content of Bla g 1 in control and exposed tubes was compared, the decrease after exposure was significant at exposure in 35 degrees C, 53 and 100% RH. In laboratory, the shortest half-life (16-38 d) of Bla g 2 was at high temperature and humidity (100% RH at 35 degrees C), whereas the longest half-life (340 d) was at 25 degrees C and 85% RH. In the apartment, the half-life was 406 d. The results indicate that Bla g 1 and Bla g 2 allergens can persist in feces for several months under usual household humidity and temperature.

IgE Binding Epitopes of Bla g 6 from German Cockroach

Bla g 6, a German cockroach allergen, shows homology to muscle protein troponin C. It contains four calcium-binding domains at amino acid (aa) residues 20-30, 56-67, 96-107, and 132-143, and its immunoglobulin E (IgE) reactivity is dependent upon calcium ion level. However, the IgE binding epitopes of Bla g 6 have not been investigated. This study aimed to analyze the IgE binding epitopes from the five peptide fragments of Bla g 6. The full-length of three Bla g 6 isoallergens (Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301) and five peptide fragments (P1: aa 1-111, P2: aa 1-95, P3: aa 33-111, P4: aa 80-151, and P5: aa 33-151) of Bla g 6.0101 were generated by polymerase chain reaction (PCR) and expressed in Escherichia coli. Enzyme-linked immunosorbent assay (ELISA) was performed on 24 patients' sera that adjusted the final concentration 10 mM of CaCl(2) to determine the IgE activities of Bla g 6. Eight sera (33.3%), 9 sera (37.5%), and 11 sera (45.8%) showed IgE reactivity to Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, respectively. Among the sera from the positive IgE reactivity, three patients' sera were selected and the IgE reactivity was measured by ELISA with the five peptide fragments of Bla g 6. Based on IgE responses, one patient's serum exhibited the strongest IgE reactivity. We assumed that the aa between 96-151 residues, including the calcium binding domains III and IV, would be important for IgE binding. These results may provide information that will yield safe diagnostic methods and immunotherapeutics.

IgE-Binding Epitope Analysis of Bla g 5, the German Cockroach Allergen

Cockroach infestations have been linked with allergic diseases such as asthma in humans. Bla g 5, sigma class glutathione S-transferase (GST), is the major cockroach allergen which has the highest IgE response value of all cockroach allergens. Although several cockroach allergens have been identified and cloned, information regarding their B ell and T cell IgE-binding epitopes is limited. In order to analyze the IgE binding epitopes of Bla g 5, full-length and five peptide fragments (A, 1-100 amino acid residue; B, 91-200; Ba, 1-125; Bb, 1-150; Bc, 1-175) were expressed. Twelve (37.5%) of 32 sera from cockroach-sensitized subjects showed positive IgE reactivity to the recombinant Bla g 5 (rBla g 5). Six strong positive sera were selected for the epitope study. Recombinant proteins not containing 176-200 amino acid residues were unable to react to sera from cockroach sensitized individuals, suggesting that this region contains the IgE-binding epitope. Despite strong IgE reactivity to rBla g 5, the pooled serum from 5 cockroach-sensitized patients did not show IgE reactivity to all synthetic peptides consisting of 15 residues covering 161-200 amino acids. These results suggest the possibility that Bla g 5 may have a conformational epitope in the C-terminal region. GST is the important target for the development of vaccines and drugs against allergic diseases because of high cross-reactivity among insect species. This study will aid recombinant allergen research for immunotherapy of cockroach allergens and other insect allergens.

Oral Tolerance Reduces Pulmonary Inflammation in a Cockroach Antigen Murine Model of Allergic Asthma

Our lab previously demonstrated that German cockroach allergens (CRA) induce allergic‐type airways inflammation in outbred (ICR:CD‐1) mice which closely resembles human allergic asthma. We determined the efficacy of oral tolerization in alleviating asthmatic symptoms. Mice were given 8ug/day CRA by oral gavage for 4 days beginning with day ‐7, followed by intratracheal challenges with 4.75ug on day 0 and 2.4ug on days 14 and 21. Cytokines and cells were measured in the bronchoalveolar lavage (BAL) fluid and cytokines and myeloperoxidase activity were measured in lung homogenate. Samples were collected 4 hours post final challenge and the results compared to PBS fed, CRA challenged mice. BAL eosinophils were significantly reduced in the CRA fed mice and eosinophil specific peroxidase activity was reduced in the lung homogenate as compared to PBS fed mice. In addition, multiple respiratory factors were significantly improved in the orally tolerized mice. These results indicate that oral tolerance abrogates eosinophil infiltration into the lung space. These beneficial effects are reflected by an improvement in pulmonary health as evaluated by respiratory parameters. BAL eosinophils and eosinophil specific peroxidase activity in the lung homogenate. Respiratory parameters, respiratory rate (RR), minute ventilation (MV), peak inspiratory flow (PIF), time of expiration (Te), time of inspiration (Ti), enhanced pause (Penh). BAL Eos EPO RR MV PIF Te Ti Penh CRA Fed 43,300 0.6 273.9 84.9 5.9 0.15 0.09 2.3 PBS Fed 158,800 1.2 223.2 67.8 4.8 0.2 0.11 3.9 p value 0.007 0.02 0.01 0.01 0.002 0.04 0.002 0.03

Adenosine A2B Receptor Expression by Myeloid Cells Mediates Airway Inflammation in Asthma

Allergic‐type airway inflammation characteristic of human asthma is modeled in mice by direct inhalation of German cockroach allergens (CRA). The role of adenosine A2B receptors (A2B) in asthma was studied in mice lacking A2B (A2B KO) and myeloid specific A2B KO (Lys‐M). CRA was given intratracheally on day 0 with two pulmonary challenges on day 14 and 21. 24 hours after final challenge lungs and brochoalveolar lavage fluid (BAL) were collected.Virtually all measures of pulmonary inflammation were reduced in the A2B KO and Lys‐M compared to wild type mice. Lung homogenate (LH) eosinophil peroxidase (WT, 1.0 ± 0.2; A2B KO, 0.2 ± 0.04*; Lys‐M, 0.3 ± 0.04*; *p &lt; 0.01), but not myeloperoxidase (WT, 0.4 ± 0.1; A2B KO, 0.4 ± 0.1; Lys‐M 0.6 ± 0.1) levels were decreased. Our results demonstrate A2B expression by myeloid cells is an important contributor to the pulmonary inflammation in asthma. *LH and ^BAL cytokines ng/mL. *^p &lt; 0.05 compared to WT Parameter WT KO Lys‐M Eotaxin 1 2.6 ± 0.2 0.9 ± 0.1* 1.0 ± 0.2* Eotaxin 2 14.7 ± 1.6 2.3 ± 0.3*^ 4.8 ± 0.1*^ IFN‐γ 8.6 ± 0.9 4.1 ± 0.2* 5.6 ± 0.6* IL‐4 3.3 ± 0.3 1.8 ± 0.1* 1.8 ± 0.2* IL‐5 3.2 ± 0.3 1.3 ± 0.1* 1.8 ± 0.3* IL‐13 7.8 ± 0.9 4.1 ± 0.3* 4.3 ± 0.6* TNF‐α 5.0 ± 0.6 1.9 ± 0.4* 2.7 ± 0.2* BAL cells *p &lt; 0.05 compared to WT Parameter (x 104) WT KO Lys‐M Eosinophils 26.2 ± 5.8 1.7 ± 0.3* 1.5 ± 0.6* Macrophages 14.9 ± 1.8 9.5 ± 0.4* 9.0 ± 0.9* Lymphocytes 7.9 ± 1.3 2.0 ± 0.3* 3.4 ± 0.5* Neutrophils 55.4 ± 5.4 43.8 ± 2.4 35.4 ± 3.8* This work was supported by NIH Grant R01 ES0135283‐03